Project Summary Hearing loss caused by hair cell degeneration is one of the most widespread sensory disabilities in the world with 30% of people between ages 65 -75 being affected. In humans and mammals, hair cells are unable to regenerate, hence any damage or loss of hair cells is permanent. Therefore, there is an urgent need to develop therapeutic approaches for regenerating hair cells in humans. In contrast to mammals, hair cells in fishes, birds and amphibians regenerate following hair cell death. Very little is known about why hair cells regenerate in some vertebrates and not in others. The goal of this work is to elucidate the gene regulatory network underlying zebrafish hair cell regeneration, The results will help us identify strategies to elicit the restoration of lost hair cells in mammals. Zebrafish possess hair cells in their ears and in their skin as part of the sensory lateral line system that senses water movement. The development of zebrafish lateral line hair cells is controlled by the same signaling pathways as mammalian inner ear hair cells, however, they are easily accessible to direct observation and genetic manipulation. Interactions between Notch and Wnt signaling are crucial for maintaining a balance between progenitor cell self-renewal and differentiation. This work will expand this knowledge by determining how other signaling pathways, such as Fgf and retinoic acid (RA), which also robustly affect hair cell regeneration, fit into this network. The epistatic relationship between the Notch, Wnt, Fgf and RA pathways will be determined by combinatorial manipulation of pathways using mutants, pharmaceutical inhibitors and transgenic lines. In addition, gene interactions that regulate the balance of progenitor cell self-renewal and differentiation will be functionally interrogated at the single cell level in vivo with a combination of time-lapse and cell proliferation/cell fate analyses. An important component of this work is the expression analysis of individual support cells during regeneration and after pathway manipulations. The single cell analysis is powerful, as an unexpected diversity or mosaicism in support cells was discovered, which is masked in bulk support cell RNASeq analyses. In addition, as cells differentiate they will integrate many different signals from other cells over time. These experiments will allow the distinction of cells on different trajectories, temporally order them along those trajectories, and identify new regulatory factors controlling differentiation. The results of these aims will inform us of the gene regulatory network underlying zebrafish hair cell regeneration, which will guide the design of therapies to induce hair cell regeneration in mammals.